Anthrax is a potentially fatal human disease caused by the Gram-positive spore-forming bacterium Bacillus anthracis. Virulence factors including the toxin crucial for B. anthracis pathogenesis are under the control of the phosphorelay signal transduction pathway that regulates post-exponential gene expression and sporulation through phosphorylation of the Spo0A response regulator transcription factor. Toxin proteins are exported to the outside of the bacterial cell where they exert their lethal effect. The research in this proposal is toward understanding anthrax toxin gene regulation and toxin secretion in order to identify new therapeutic targets for intervention and persistence. Proposed is the development of new and facile methods for gene inactivation, allele exchange and regulated gene expression in B. anthracis in order to dissect and identify those genes regulating toxin synthesis. Studies are envisioned to evaluate these genes in a pathogenesis model system leading to a more complete picture of the regulation of virulence factor synthesis and pathogenesis. The sensor histidine kinases that regulate the flow of phosphoryl groups through the phosphorelay to ultimately regulate toxin synthesis will be targets of investigation. Experiments are proposed to identify the signals recognized by B. anthracis that induce the phosphorelay. Genetic and biochemical approaches to the understanding of the mechanism by which toxin protein precursors are processed and secreted through the cytoplasmic membrane are proposed. The roles of the multiple signal peptidases in the secretion of toxin and other secreted proteins will be determined. Extensive physical characterization of the AbrB transition state regulator that represses toxin production will be undertaken to decipher how it is able to recognize seemingly random but specific DNA sequences in promoters. A combined in vivo and structural approach is proposed to pinpoint sites for intervention in the function of regulatory proteins leading to the synthesis of active anti-infective agents. Initial proof of concept studies will focus on the Spo0A response regulator for which extensive structural and biochemical information exists. This is a collaborative effort between investigators with expertise in genetics, biochemistry and structure of Bacillus regulatory proteins. [unreadable] [unreadable]